Studies On Regulation Mechanisms Underlie Blind-side Hypermelanosis Of Farmed Flatfish

Blind-side hypermelanosis phenomenon in farmed flatfish, especially in native flatfish species Cynoglossus semilaevis Güntherand Paralichthys olivaceus, has become the bottleneck of sustainable development of flatfish farming industry. In order to construct practical control and regulation technology, the underlying mechanisms for this blind-side hypermelanosis should be clarified at first. In the present study, the in vitro prokaryotic expression systems of two key hyperpigmentation-inhibiting genes(MCH1, MCH2) of Cynoglossus semilaevis Günther were established by combining molecular cloning, prokaryotic expression, western blotting and in vitro incubation of pitutary tissues, the purified and biologically active recombinant proteins were obtained. In order to exploit the possible mechanisms underlie degeneration of blind-side hypermelanosis in pond-culture Japanese flounder, the morphological observation, morphometric statistical analysis, plasma hormone detection and gene expression methods were used,and the possible mechanism was revealed. Results from the present study will be helpful for better understanding of the hyperpigmentation on the blind side of half-smooth tongue sole and development of practical regulation technology. The main results are listed as follows: 1. Prokaryotic expression and bioactivity analysis of MCH1 from Cynoglossus semilaevis GüntherThe mature peptide domain of melanin concentration hormone1(MCH1)from Cynoglossus semilaevis Günther was amplified with specific primers based on their cDNA sequences from NCBI. Then the matured peptide fragments were subcloned into the prokaryotic expression vector pET-32 a to successfully construct MCH1/pET32 a recombinant plasmid. The obtained recombinant MCH1 protein expressed in form of inclusion bodies with molecular weight of 29.9 KD and maximally accounted for 50% of the whole bacterial protein post 6-hour induction with 0.2 mmol/L IPTG at 32℃. Western blotting analysis indicated that the obtained recombinant MCH1 protein had the antigenicity to His 6 antibody.The obtained target MCH1 protein was purified using Ni2+-NTA affinity chromatography method. The bioactivity of obtained target MCH1 protein was tested by the method of in vitro incubation of pituitary gland. The results showed that MCH1 recombinant protein can effectively stimulate or inhibit the secretion of pituitary MCH and MSH peptides. Wherein, the MCH peptide level increased with the MCH1 protein concentration and peaked at 100 nmol/L, however it dramatically decreased at 1000nmol/L; the MSH peptide level showed a significant rise at 100nmol/L group, and peaked at 1000nmol/L of MCHI1 protein. As for gene expression patterns, the MCH1 and MCH2 mRNAs levels showed similar variation trend, wherein MCH1 mRNA showed upregulated expression with the recombinant MCH1 protein increased, and peaked at 10 nmol/L group, and MCH2 mRNA levels peaked at 100 nmol/L group. The pituitary PACAP and POMC-a mRNAs both exhibited down-regulated expression trends, and POMC-b, MCHR1, MCHR2, MITF mRNAs expression levels were not significantly affected by exogenous recombinant MCH1. In summary, the recombinant MCH1 protein could adjust the physiological functions of pituitary hormones secretion and genes expression. The present results could provide basic knowledge and technical support for development of high-efficient and specific additives which would be applied into aquaculture of Cynoglossus semilaevis. 2. Prokaryotic expression and bioactivity analysis of MCH2 from Cynoglossus semilaevis GüntherThe mature peptide domain of melanin concentration hormone2(MCH2)from Cynoglossus semilaevisGünther was amplified and subcloned into the prokaryotic expression vector pET-32 a to successfully construct MCH2/pET32 a recombinant plasmid. The obtained recombinant MCH1 protein expressed in form of inclusion bodies with molecular weight of 32.1 KD and maximally accounted for 55% of the whole bacterial protein post 6-hour induction with 0.2 mmol/L IPTG at 27℃. Western blotting analysis indicated that the obtained recombinant MCH2 protein had the antigenicity to His6 antibody.The obtained target recombinant MCH2 protein was purified using Ni2+-NTA affinity chromatography method. The bioactivity of obtained target MCH2 protein was tested by in vitro incubation of pituitary gland method. The results showed that recombinant MCH2 protein can effectively stimulate or inhibit the secretion of pituitary MCH and MSH peptides. Wherein, the MCH peptide level increased with the MCH2 protein concentration and peaked at 1000 nmol/L; the MSH peptide level showed a significant rise at 1000 nmol/L group. As for gene expression patterns, the MCH1 and MCH2 mRNAs levels showed similar variation trend post recombinant MCH2 protein treatment. The expression of pituitary PACAP and POMC-a mRNAs both exhibited up-regulated expression trend, and MCHR1, MCHR2, MITF mRNAs expression levels were not significantly affected by exogenous recombinant MCH2 protein. In summary, the recombinant MCH2 protein could adjust the physiological functions of pituitary hormones secretion and gene expression. The present results could provide basic knowledge and technical support for development of high-efficient and specific additives which could be applied into the blind-side hypermelanosis control in Cynoglossus semilaevis farming industry. 3. Physiological mechanisms for fading of blind-side hypermelanosis in pond-culture Paralichthys olivaceusThe blind-side hypermelanosis fading of pond-culture Japanese flounder Paralichthys olivaceus was observed, the possible mechanism was investigated by using morphological observation, morphometric statistical analysis, plasma hormone detection and gene expression methods. The number of melanocytes in fading area of blind side is significantly less than that of eye-side area and blind-side hypermelanosis area. Moreover, the scales in blind-side fading area experienced the transformation from ctenoid scale to →weak ctenoid →cycloid scales. Meanwhile, the number of ctenoid scale spines decreased. The plasma MSH and MCH levels were determined and compared between three types of Japanese flounder including blind-side hypermelanosis type(BSH), normal blind side type(NBS) and blind-side hypermelanosis fading type(BSD). The results showed that the plasma MCH level in BSD fish was significantly higher than that of NBS and BSH fish. Whereas, the BSH fish had the highest MSH level among the three types. Gene expression analysis showed that the pituitary MCH mRNA level in BSD fish was remarkably higher than that of BSH, but for POMC mRNA, the BSD fish showed significant lower expression level. More important, the brain POMC1 mRNA level in BSD fish was significantly lower than BSH fish and NBS fish. Results from the present study would be helpful for better understanding of the mechanisms underlie blind-side hyperpigmentation of half-smooth tongue sole, and would be beneficial for development of practical regulation technology for blind-side hypermelanosis.